The ease with which small countries and terrorist groups can now obtain biological warfare agents has escalated the need to provide the war fighter and civilians alike with miniature, easy to use, disposable instruments for detection and identification of potentially hazardous biological agents (Iqbal et al. Biosensors & Bioelectronics 2000, 15, 549–578; Christel, L. A., et al. J. Biomech. Eng. 1999, 121, 22–27; Higgins, J. A., et al. Ann. NY Acad. Sci. 1999, 894, 130–148; and Hood, E. Environ. Health Perspect. 1999, 107, 931–932). Traditional methods for detection and identification of microorganisms, viruses and/or their products lack the speed and sensitivity to be of field usage since they are not real time or even typically completed in a single day. Microbial and viral identification assays have as their basis, the principle that dates back to the days of Pasteur, i.e. the growth of the organism in culture or replication of virus in a suitable host (Reischl, U., Frontiers Biosci., 1996, 1, Application of molecular biology-based methods to the diagnosis of infectious diseases. 1, e72–e77). Toxin identification has typically relied on biological assays, which although relatively rapid, usually requires purification of the toxin prior to testing (Feng, P. Mol. Biotechnol. 1997, 7, 267–278; van der Zee, H. et al. J. AOAC Int. 1997, 80, 934–940). Depending upon the nature of the agent to be detected, this process can take from days to months (Pillai, S. D. Arch. Virol. 1997, 13 Suppl., 67–82; van der Zee, H. et al. J. AOAC Int. 1997, 80, 934–940). Clearly, this is not practical in situations where detection and identification may be required for protecting a population from hazardous biological agents. Molecular recognition systems that can be used for rapid identification can improve response time and thus avert or reduce the number of casualties associated with a potential bioterrorism or biowarfare event.
Biological threat agents can be either infectious or toxigenic organisms or simply toxins (Hood, E. Environ. Health Perspect. 1999, 107, 931–932; Haines, J. D. et al J. Okla. State Med. Assoc. 1999, 93, 187–196). Examples of the former are Bacillus anthracis(anthrax) and Yersinia pestis (plague) while the latter is exemplified by staphylococcal enterotoxin B or botulinum toxin. Detection of toxins has followed a similar track as that observed for detection of chemical threat agents. Typically, toxins are detected on the basis of their respective chemical structures. Detection of mycotoxins has been accomplished using traditional analytical chemistry tools such as gas chromatography-mass spectrometry (GC-MS) (Black, R. M et al. J. Chromatogr. 1986, 367, 103–116). Although precise and highly sensitive, GC-MS does not lend itself to field applications and cannot be easily applied to complex target analytes such as bacteria. Specific compounds, i.e. signature components might be identified in targeted bacterial agents but this approach tends to be too complex for routine, high throughput analysis (van der Zee, H. et al. J. AOAC Int. 1997, 80, 934–940; Hood, E. Environ. Health Perspect. 1999, 107, 931–932).
A number of technological innovations have provided tools that have made detection and identification of microorganisms, viruses and their products faster and more sensitive. Two significant technologies that have had dramatic impact on potentially rapid detection are: (1) generation of monoclonal antibodies; and (2) collection of individual methodological advances that have formed the basis of recombinant DNA technology. A key event in the latter technology is the development of polymerase chain reaction (PCR) technology which as a singular process, has significantly reshaped the authors' thinking with respect to detection of biological agents.
Identification of biological threat agents involves recognition of bacteria (vegetative cells and spores), viruses and toxins. Nucleic acid and immunology-based methods for identification of bacteria, viruses and their products (antigens and toxins) have found wide application including testing of food, clinical and environmental samples. Extension of these applications for detection and identification of biological threat agents is reasonable since basic principles involved are identical. Two essential features characterize these applications, i.e. the need to: (1) develop a target specific identification method; and (2) formulate an assay that will work on the requisite sample. However, the majority of the methods developed meet the first criterion, i.e. identify the target organism or toxin, but fail to be sufficiently robust to work on “real” samples.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. Low-resolution MS may be unreliable when used to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to detect a particular organism.
Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
Several groups have described detection of PCR products using high resolution electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., J. Am. Soc. Mass Spec., 1996, 7, 1266–1269; Muddiman et al., Anal. Chem., 1997, 69, 1543–1549; Wunschel et al., Anal. Chem., 1998, 70, 1203–1207; Muddiman et al., Rev. Anal. Chem., 1998, 17, 1–68). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 1996, 10, 377–382). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec., 1999, 13, 1201–1204). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
Thus, there is a need for methods for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need as well as other needs.